The human liver is the primary site for detoxification of ingested chemicals and also facilitates the metabolic breakdown of a wide range of pharmaceutical compounds. Primary hepatocytes, isolated from liver tissue and subsequently cultured, can be used to test the metabolic breakdown and toxicity of various compounds. However, primary hepatocytes do not divide, and when cultured, rapidly lose their ability to produce functional hepatic enzymes. In addition, inherent variability between different preparations of primary hepatocytes complicates comparisons of results obtained from different primary hepatocyte cultures.
Although it has been suggested that human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) could be differentiated to mature hepatocytes suitable for these purposes, cells isolated to date from these pluripotent cells are fetal, or immature, in nature, notwithstanding their hepatocyte-like morphology. As a result, they produce functional hepatic enzymes needed for testing purposes in very small quantities, if at all.
Accordingly, it is recognized in the art that the term “hepatocyte” should not be broadly applied to include stem cell-derived fetal hepatocytes that express some markers expressed by primary hepatocytes, unless the cells also produce functional hepatic enzymes in quantities comparable to the quantities produced by primary hepatocytes. For example, Hengstler et al. (Expert Opin. Drug Metab. Toxicol. (2005) 1(1): 61-74) state that the term “hepatocyte” should only be used to define stem cell-derived cells that both express known hepatocyte markers and produce quantities of functional hepatic enzymes that are comparable to the quantities produced by primary hepatocytes (page 63). Furthermore Hengstler et al. explain that a “hepatocyte” should exhibit drug metabolism capabilities and should be capable of generating toxic metabolites as human primary hepatocytes would (page 62, col. 2). Finally, Hengstler et al. indicate that qualitative assays like reverse transcription PCR and immunochemical staining are not sufficient to establish that cells are hepatocytes, and that quantitative assays that include human hepatocyte controls are required to demonstrate the generation of true hepatocytes (page 63, col. 2; page 71). As another example, Soto-Gutierrez et al. (Biotechnology and Genetic Engineering Reviews (2008) 25: 149-164) emphasize that a “hepatocyte” should be defined to encompass only cells that are able to perform the functions of primary hepatocytes, including metabolizing xenobiotics or other endogenous substances (page 155).
Cai et al. previously reported the differentiation of human embryonic stem cells into hepatic cells (Hepatology (2007) 45: 1229-1239). Although Cai et al. purportedly show the expression of some functional genes, they presented no confirmatory quantitative or drug metabolism data. The expression of functional genes was demonstrated by reverse transcription PCR and immunochemical staining, neither of which is quantitative. In these assays, minute quantities of RNA and protein, respectively, can result in a positive test, and there is no accurate indication of the quantity of gene expression occurring in the cell. Furthermore, no comparison is made to results obtained using human hepatocyte controls. As discussed above, these results are insufficient to establish the formation of true hepatocytes.
When the inventors used a protocol similar to that described by Cai et al., they obtained cells having hepatocyte morphology and expressing some genes characteristic of hepatocytes. However, when such gene expression was quantitatively measured and compared to that of primary human hepatocytes isolated from liver tissue, the expression levels were found to be too low to label the resulting cells as true hepatocytes. Thus, these cells are more properly considered fetal hepatocytes, not true hepatocytes.
Takayama et al. recently reported the production of functional hepatocytes from human embryonic stem cells and human induced pluripotent stem cells using a protocol similar to that used by Cai et al., but additionally including the sequential transduction of three separate factors: SOX17, HEX, and HNF4α (Molecular Therapy (8 Nov. 2011); doi:10.1038/mt. 2011.234: 1-11). Because the Takayama protocol requires three separate transductions, it would be too complex and time consuming to readily put into practice on a larger scale. Furthermore, improved enzyme induction with known inducing agents and higher levels of CYP enzyme expression than is reported by Takayama et al. would be desired in hepatocytes used for metabolism or toxicity testing.
Thus, there is a need in the art for an improved, relatively simple, replicable and standardized method of producing mature hepatocyte cultures that have the ability to produce functional hepatic enzymes for an extended period of time, for use in toxicity and metabolism testing.